Phospholipids from herring roe improve plasma lipids and glucose tolerance in healthy,

young adults
Bodil Bjrndal1*, Elin Strand1, Jennifer Gjerde12, Pavol Bohov1, Asbjrn Svardal1,Bernd
WK Diehl4, Sheila M Innis3, Alvin Berger56 and Rolf K Berge17

*Corresponding author: Bodil Bjrndal bodil.bjorndal@k2.uib.no

Author Affiliations
1

Department of Clinical Science, University of Bergen, Bergen N-5020, Norway

Hormonlaboratoriet, Haukeland University Hospital, Bergen N-5021, Norway

Department of Paediatrics, University of British Columbia, Vancouver, BC V5Z4H4,

Canada
4

Spectral Service AG, Kln D-50996, Germany

Arctic Nutrition AS, rsta N-6155, Norway

Department of Food Science & Nutrition, University of Minnesota, St. Paul, MN 55108-

1038, USA
7

Department of Heart Disease, Haukeland University Hospital, Bergen N-5021, Norway

For all author emails, please log on.

Lipids in Health and Disease 2014, 13:82 doi:10.1186/1476-511X-13-82

The electronic version of this article is the complete one and can be found online
at:http://www.lipidworld.com/content/13/1/82
Received:
Accepted:
Published:

13 March 2014
10 May 2014
17 May 2014

2014 Bjrndal et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited.
The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.

Abstract
Background
Herring roe is an underutilized source of n-3 polyunsaturated fatty acids (PUFAs) for human
consumption with high phospholipid (PL) content. Studies have shown that PL may improve
bioavailability of n-3 PUFAs. Arctic Nutritions herring roe product MOPL30 is a PL:
docosahexaenoic acid (DHA)-rich fish oil mixture, with a DHA:eicosapentaenoic acid (EPA)
ratio of about 3:1, which is also rich in choline. In this pilot study, we determined if MOPL30
could favorably affect plasma lipid parameters and glucose tolerance in healthy young adults.
Methods
Twenty female and one male adults, between 22 and 26 years of age, participated in the study.
Participants took encapsulated MOPL30, 2.4 g/d EPA + DHA, for 14 days, and completed a
three-day weighed food record before and during the capsule intake. Plasma lipids and their fatty
acid (FA) composition, plasma and red blood cell (RBC) phosphatidylcholine (PC) FA
composition, acylcarnitines, choline, betaine and insulin were measured before and after
supplementation (n=21), and one and four weeks after discontinuation of supplementation
(n=14). An oral glucose tolerance test was performed before and after supplementation.
Results
Fasting plasma triacylglycerol and non-esterified fatty acids decreased and HDL-cholesterol
increased after 14 days of MOPL30 intake (p<0.05). The dietary records showed that PUFA
intake prior to and during capsule intake was not different. Fasting plasma glucose was
unchanged from before to after supplementation. However, during oral glucose tolerance testing,
blood glucose at both 10 and 120 min was significantly lower after supplementation with
MOPL30 compared to baseline measurements. Plasma free choline and betaine were increased,
and the n-6/n-3 polyunsaturated (PUFA) ratio in plasma and RBC PC were decreased postsupplementation. Four weeks after discontinuation of MOPL30, most parameters had returned to
baseline, but a delayed effect was observed on n-6 PUFAs.
Conclusions
Herring roe rich in PL improved the plasma lipid profile and glycemic control in young adults
with an overall healthy lifestyle.
Keywords:
Herring roe; Phospholipids; Eicosapentaenoic acid; Docosahexaenoic acid; Omega-3
polyunsaturated fatty acids; Glycemic control; Choline; Acylcarnitines
Background
The health benefits of a higher fish intake, thereby increasing the intake of n-3 long-chain
polyunsaturated fatty acids (PUFAs) and reducing the n-6 PUFA/n-3 PUFA ratio, has been
documented in several studies [1]. Cardioprotective effects of n-3 PUFAs, in particular
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been attributed to reduction
in fasting triacylglycerol (TAG), blood pressure lowering, anti-inflammatory and antiarrhythmic
effects, improved insulin sensitivity and vascular endothelial function, and reduced thrombotic

tendency [2]. The efficacy of n-3 PUFAs in the prevention of heart disease has been challenged
in recent meta-studies, but it is important to note that newer studies could be hampered by a
higher general intake of n-3 PUFAs and improved treatment protocols for heart patients [3]. The
current recommended intake is 250 mg/day EPA + DHA for the general population, and
300 mg/day for pregnant women (European Food Safety Authority (EFSA)). The American
Heart Association (AHA) recommends 1 g/day EPA + DHA for patients with cardiovascular
disease (CVD). Combined with the increased focus on n-3 PUFA intake in the media, this has
led to a large n-3 PUFA supplement market dominated by fish oil from sardines. However, the
need for new sources of high-quality EPA and DHA is increasing.
Immature roe from spring-spawning Norwegian herring is an underutilized source of n-3 PUFArich phospholipids (PLs). Of the approximately 600,000-ton herring caught in Norway each year,
only a small percentage of herring roe is used for human consumption. The product MOPL30
(Arctic Nutrition) from herring roe contains about 45% n-3 PUFA (mg/g product basis), with a
DHA:EPA ratio of approximately 3:1. In addition, 30% of the lipids are PL of which most (75%)
in the form of phosphatidylcholine (PC). Thus herring roe provides choline, an important
nutrient involved in many biochemical pathways.
The bioactivity of n-3 PUFAs may be influenced by the lipid structures in which they are
incorporated. PLs and free fatty acids have increased bioavailability compared to TAG and ethyl
ester forms of n-3 PUFAs, respectively [4,5]. PL from krill have been shown to influence gene
expression more than TAG from fish oil in mice, at a similar dose of EPA and DHA [6]. In
particular, genes involved in glucose and lipid metabolism were more affected by PL than
TAG[6,7]. A recent study comparing the bioavailability of PL and TAG in the form of krill oil
and fish oil in healthy subjects demonstrated increased levels of EPA and DHA in plasma and
red blood cells (RBC) after four weeks of krill oil intake compared to fish oil [8]. In addition,
animal studies have shown a more efficient reduction in plasma lipid levels with PL compared to
TAG intervention [9]. Herring roe oil supplementation has shown promising results in animal
studies, including reduction in plasma lipids and inflammatory parameters, and improved insulin
sensitivity [10,11].
The aim of this study was to determine the effect of n-3 PUFA when given in PL-rich herring roe
on blood lipids and glucose tolerance in healthy subjects with a balanced diet, and to determine
uptake of n-3 PUFAs into red blood cells as a measure of bioavailability. Follow-up samples
were included to determine how long the n-3 PUFA remained in circulation after discontinuation
of supplementation (washout effects).
Results
Characteristics of the study population
The study included twenty-one young, healthy individuals aged 20 to 26 years, with a mean
SD body mass index of 21.22.8, and range between 15.1 and 26.7. Fourteen of the participants
completed a three day weighed food record during the two weeks prior to supplementation, and
again during the two weeks intervention. The energy % from PUFA in the habitual diet was
(mean SD) 7.52.8 before and 6.72.0 during the intervention (p=0.264, n=14), indicating
that the dietary intake of PUFA remained unchanged during the study period. Furthermore, there
was no change in dietary intake of total fat, saturated fatty acids (FA), monounsaturated FA,
protein or carbohydrate (data not shown).

The capsules were taken during a meal, and there were no reports of reflux or unpleasant taste
following capsule intake.
MOPL30 supplementation affected plasma lipid levels
Plasma lipid levels were measured at baseline (start) and after (end) two weeks of MOPL30
supplementation. In addition, washout (WO) samples were taken one and four weeks after the
final day of capsule intake. The TAG level was significantly reduced in the end samples (21%
reduction). However, the reduction at WO week one (p=0.10) and four (p=0.30) compared to
start was not significant (Figure 1a). Non-esterified FAs (NEFAs) were also significantly
reduced after two weeks of MOPL30 intake (23.3%), and remained unchanged, however
insignificantly, one (34.3%, p=0.17) and four weeks (29.8%, p=0,24) after discontinued
supplementation (Figure 1b). Despite a high PL content in the supplement, plasma PL levels
were not altered (Figure 1c).
Figure 1. Plasma lipid levels in response to MOPL30. Levels of triacylglycerol
(TAG, a), non-esterified fatty acids (NEFA, b), phospholipids (c), total cholesterol (d), highdensity lipoprotein (HDL) cholesterol (e), low-density lipoprotein (LDL) cholesterol (f), and the
HDL/LDL cholesterol ratio (g) in fasted plasma samples taken at baseline (start), after 14 days of
supplement (end), and one week (wash out (WO) week 1) and four weeks (WO week 4) after
discontinuation of supplement. Values are given as means with standard deviations, and
significant changes between start and end (n=21) and start and WO (n=13) are indicated
(*p<0.05, **p<0.01, ***p<0.001).
The total cholesterol level in plasma was not changed by MOPL30 intake (Figure 1d). HDLcholesterol, however, increased by 5.5% at end compared to start (Figure 1e), while LDLcholesterol was unchanged (Figure 1f). This led to a trend towards an increased HDL/LDLcholesterol after MOPL30 intake (9.0%; Figure 1g). Surprisingly, while plasma HDL-cholesterol
returned to initial values after four weeks of WO, total- and LDL-cholesterol was significantly
reduced at 4 week WO compared to start samples (10.6% reduction in WO week 4 vs. start).
Fatty acid composition in plasma and red blood cells
Total fatty acid composition was measured in plasma before and after two weeks of supplement.
Total n-3 PUFAs in plasma were increased 1.6 fold, mainly due to a 2.2 fold increase in EPA,
and a 1.5 fold increase in DHA (Table 1). One week after the end of supplementation, EPA and
DHA had decreased by 44% and 24%, respectively, compared to the end values. By four weeks
of WO, all plasma n-3 PUFAs had returned to start levels. Concomitant with the increase in EPA
and DHA during the 2 weeks supplementation, n-6 PUFAs showed a decrease of 8.2%.
Interestingly, n-6 PUFAs, in particular arachidonic acid (AA) did not return to start levels as
quickly as EPA and DHA after discontinuation of supplementation.
Table 1. Plasma fatty acids during the study
Rather AA remained lower than start values in the four-week WO samples. Although mead acid
was low in start samples, as expected with a high habitual PUFA intake, it still showed a
significant decrease with the MOPL30 supplement (27.7% reduction). The total fatty acid

content in plasma was significantly reduced after two weeks of supplement, consistent with the
reduced TAG and NEFA. The n-6/n-3 PUFA ratio was within recommendations in the start
samples, consistent with the participants balanced intake of fatty acids (Table 1). The n-6/n-3
ratio was further decreased by 44.8% post-supplementation, but had returned to start values at
WO week 4.
As most of the EPA and DHA in MOPL30 are in the form of PC, the fatty acid composition of
PC was measured both in plasma and in red blood cells. The findings in plasma PC paralleled the
findings in total plasma FAs, with a 2.1 fold increase in EPA and a 1.6 increase in DHA postsupplementation, resulting in a 45.2% decrease in the n-6/n-3 PUFA ratio (Table 2). At WO
week 1, EPA and DHA were reduced by 39.3% and 21.9%, respectively, relative to end values.
Incorporation of EPA and DHA into RBC membranes is believed to better indicate long term
storage than plasma levels [12]. We found a 2.1 fold increase in EPA and a 1.4 fold increase in
DHA post-supplement, similar to results in plasma, resulting in a 1.7 fold increase in the RBC
PC omega-3 index and a 40.6% reduction in the n-6/n-3 PUFA ratio (Table 3). AA in the plasma
PC and RBC PC was not significantly altered by MOPL30, although it was significantly reduced
in plasma total fatty acids (Tables 1, 2 and 3). The WO effect on the RBC PC EPA was similar to
that in the plasma PC after one week (39% reduction relative to end values), while the decrease
in the RBC PC DHA was lower (11% reduction relative to end values).
Table 2. Plasma phosphatidylcholine fatty acids during the study
Table 3. Red blood cell phosphatidylcholine fatty acids during the study
Plasma choline increased with MOPL30
Both choline and its metabolite betaine increased in plasma after two weeks of MOPL30
supplement (Figure 2). Although the intake of PC was increased during supplementation, total
plasma- and RBC PCs were reduced post-supplementation, as measured by 31P nuclear magnetic
resonance (NMR) (Figure 3a and b). However, a higher proportion of EPA and DHA-containing
PCs were observed in both plasma and RBC (Tables 2 and 3). Plasma TAG was reduced, while
no change was seen in cholesterol esters (Figure 3a). Total cholesterol, measured by 1H NMR,
was not influenced by two weeks of supplement (mean SD start vs end; 0.950.18 mmol/L vs
0.880.019 mmol/L, p=0.096). The plasma TAG level measured with NMR was similar to the
plasma TAG-level measured by enzymatic-analysis (Figure 1). Sphingomyelin (SPH), PC, and
cholesterol were reduced in RBC by two weeks of MOPL30 treatment (Figure 3b). Interestingly,
while cholesterol returned to start levels after four weeks WO, SPH and PC remained lower than
start values.
Figure 2. Plasma choline and betaine levels in response to MOPL30. Levels
of choline (a) and betaine (b) in fasted plasma samples taken at baseline (start), after 14 days of
supplement (end), and one week (wash out (WO) week 1) and four weeks (WO week 4) after
discontinuation of supplement. Values are given as means with standard deviations, and
significant changes between start and end (n=21) and start and WO (n=13) are indicated
(*p<0.05, **p<0.01, ***p<0.001).

Figure 3. Lipid classes in plasma and red blood cells (RBC) in response to
MOPL30. Comparison of the lipid classes sphingomyelin (SPH), triacylglycerol (TAG),
phosphatidyl ethanolamine (PE), lysophosphatidylethanolamine (LPC), phosphatidyl choline
(PC), and cholesterol esters (CE) at baseline (start) and after 14 days of supplement (end) in
fasting plasma samples (a). Comparison of the lipid classes SPH, TAG, and cholesterol (Chol.)
at start, end, and one week (wash out (WO) week 1) and four weeks (WO week 4) after
discontinuation of supplement in RBC (b). Analysis was done by 31P NMR. Values are given as
means (%w/w=g/100 g plasma) with standard deviations, and significant changes between start
and end (n=21) and start and WO (n=13) are indicated (*p<0.05, **p<0.01).
Plasma carnitine and acylcarnitine levels
Carnitine is essential in the transport of long-chain fatty acids across the mitochondrial
membranes. High serum levels of long- and medium-chain plasma acylcarnitines are linked to
increased risk of disease progression in patients with cardiac disease, and may indicate defects in
mitochondrial function [13]. In the current study with healthy individuals, carnitine was
insignificantly reduced (p=0.054), while its precursors -butyrobetaine and trimethyllysine were
reduced by MOPL30 (Figure 4a-c). In addition, all measured plasma acylcarnitines except the
medium-chain octanoylcarnitine (8-carbon, p=0.103), were significantly reduced by MOPL30
(Figure 4d-h). The largest reduction (29% compared to start) was seen for the short-chain
acetylcarnitine (2-carbon). The effect on -butyrobetaine, trimethyllysine and all acylcarnitines
remained significantly reduced compared to start in both the one- and four week WO samples,
indicating a possible prolonged effect of the supplement on these parameters.

Figure 4. Plasma carnitine and acylcarnitines in response to

MOPL30. Levels of carnitine (a) and the carnitine precursors gamma-butyrobetaine (b),
trimethylysine (c), even-chain acylcarnitines propionylcarnitine (d), and
isovaleryl/valerylcarnitine (e), and odd-chain acylcarnitines acetylcarnitine (f),
octanoylcarnitine (g), and palmitoylcarnitine (h) in fasted plasma samples taken at baseline
(start), after 14 days of supplement (end), and one week (wash out (WO) week 1) and four weeks
(WO week 4) after discontinuation of supplement. Number of carbons in the acyl-chains are
indicated by C2-16. Values are given as means with standard deviations, and significant changes
between start and end (n=21) and start and WO (n=13) are indicated (*p<0.05, **p<0.01,
***p<0.001).
Oral glucose tolerance test

Since previous studies in animals have indicated improved glucose metabolism after herring roe
diets, we investigated the effect of two weeks MOPL30 supplement on glucose tolerance in
healthy individuals. No change was observed in fasting insulin and glucose before and after the
supplementation period (Figure 5a and b). However, the blood glucose level in response to a 75 g
oral dose of glucose was reduced both at 10 and 120 minutes post-ingestion after the intervention
(Figure 5c). Although all participants were individuals with normal glucose sensitivity according
to their glucose tolerance test results, the area under the curve was significantly decreased,
suggesting improved glucose response (Figure 5d).

Figure 5. Fasting glucose and insulin levels, and oral glucose

tolerance. Levels of glucose (a), insulin (b), in fasted plasma and serum samples, respectively,
taken at baseline (start), after 14 days of supplement (end), and one week (wash out (WO) week
1) and four weeks (WO week 4) after discontinuation of supplement. Significant changes
between start and end (n=21) and start and WO (n=13) are indicated by P-values. Oral glucose
toleranse was measured at start and end of the experiment, blood glucose levels at baseline, 10,
30, 60, and 120 minutes after glucose ingestion are given (c). The area under the curve was
calculated at start and end (d). All values are given as means with standard deviations, and
significant changes between start and end (n=21) are indicated by *p<0.05.
Discussion
There is an increased demand for new sources of high-quality n-3 PUFAs for human
consumption, and PC-rich lipids from herring roe is a promising product in this regard.
Supplementation with DHA- and EPA-rich PC may have additional benefits compared to DHAand EPA-rich TAG both due to PL being more easily incorporated into cellular membranes, as
well as being a source of the essential nutrient choline. In this two-week intervention in young
healthy adults with high habitual fish intakes, there was a rapid increase in EPA and DHA in
RBC PC, plasma FA and plasma PC. Furthermore, there was a corresponding improved lipid
status, including a decrease in plasma TAG and NEFA, and increased HDL-cholesterol, choline,
and betaine. These findings demonstrate that MOPL30 had significant biological effects in
healthy subjects.
The participants were given 2350 mg EPA and DHA daily, comparable to doses utilized in
patients with CVD or metabolic syndrome to achieve a TAG-lowering effect. In line with this,
plasma TAG was reduced and HDL-cholesterol increased after only two weeks of treatment.
This indicates a high bioavailability of MOPL30 and a subsequent rapid effect on lipid
metabolism. It has been shown that the preferred lipid form for transport of DHA to RBC is lysoPC, which is rapidly converted to PC [14,15]. This could mean that DHA-rich PC may be
preferred for uptake in RBC and putatively in brain. Incorporation of EPA from fish oil into
RBC membranes has been shown to reach a steady state after 180 days [12]. Notably, we
observed an increase in EPA and DHA in RBC PC similar to that of plasma after only 14 days.

In mice, we recently showed that similar amounts of EPA and DHA in liver PL were achieved
with krill oil and a two-fold higher dose of EPA and DHA from fish oil, indicating higher
bioavailability of EPA and DHA from the PL-source krill oil[16]. Importantly, in a recent study
in which subjects were supplemented with 600 mg EPA and DHA from either fish- or krill oil,
the omega-3 index was significantly higher in subjects receiving krill oil than in those given fish
oil [8]. However, no effect on plasma TAG was observed at 600 mg/day EPA + DHA. A recent
study in adults with high TAG levels showed that a dose of 0.5-2 g/day krill oil for 12 weeks
significantly reduced TAG [17].
Although the intake of DHA exceeded that of EPA by 2.8 fold, the increase in EPA was higher
than DHA in the PC-fraction of both plasma and red blood cells after two weeks of MOPL30.
Studies have shown that the DHA level in lipid pools has a less steep doseresponse curve than
EPA, which is easy to influence by supplementation [18], and our results confirm this. This can
partly be due to more efficient liberation of DHA from chylomicrons [19], leaving more EPA in
chylomicron remnants and hereby making more EPA available for PL synthesis in liver. In
addition, retro-conversion of DHA to EPA is dependent on peroxisomal -oxidation, and is
reported to be at approximately 10% in humans [20,21]. Hansen et al. showed that 4 g
supplement of pure EPA for 5 weeks led to a 6.2 fold increase in EPA in plasma phospholipids,
and no change in DHA [18]. In contrast, the same intervention using a pure DHA supplement led
to a 1.9 fold increase in DHA and a 1.7 fold increase in EPA. This demonstrates the importance
of supplementation with DHA and not only EPA. Despite a dose of 132 mg DPA/day, and the
possibility of formation of DPA from EPA, DPA levels were not influenced by two weeks of
MOPL30. In line with this, a decrease in DPA after uptake of EPA or DHA has been reported in
long-term studies [22,23].
Indications of differential effects of n-3 PUFA in PL and TAG form have also been found at the
gene level in several animal studies [6,7], including genes involved in glucose metabolism. A
recent study reported that EPA and DHA supplements may improve insulin sensitivity in young
obese individuals [24]. While some meta-studies have failed to show an effect between n-3
PUFA intake and incident type 2 diabetes mellitus (T2DM) [25,26], others indicate a reduced
risk of T2DM with increased intake of PUFAs [27,28]. Based on findings in humans and from
recent animal studies, PL supplements could be expected to have potent effects on glucose
metabolism. The small, but significant, improvement in glucose response in healthy individuals
after only two weeks of intervention suggests a potential for the use of MOPL30 in insulin
resistant individuals, or patients with diabetes.
The conditionally essential nutrient choline is a quaternary amine, and is mainly utilized for the
synthesis of PC and sphingomyelin, as well as lysophosphatidylcholine. In addition, choline can
be oxidized to betaine, which is involved in the remethylation of homocysteine to methionine in
the one-carbon cycle [29]. Finally, in the neurons choline is a precursor for the important
neurotransmittor and vasodilator acetylcholine [30-32], and increased intake has been connected
with improved cognition, learning and memory [33-35]. Betaine holds an important role in the
liver, and has a potential therapeutic use in the treatment of fatty liver disease as well as
homocysteinemia, a risk factor for CVD [36,37]. In addition, some studies have demonstrated
that betaine supplements improve muscle performance [38]. PC biosynthesis is required for
VLDL production, both through the CDP-choline (Kennedy) pathway and the
phosphatidylethanolamine N-methyltransferase (PEMT) pathway. In general, a balanced diet
will provide sufficient amounts of choline, but groups which may benefit from choline
supplementation are pregnant and lactating women, infants, and cirrhosis patients [39]. Thus, a

PC supplement can both reduce the need for methyl-donors for PC synthesis, and supply betaine
for homocysteine remethylation, which will be beneficial in situations where methyl donors are
limited [40]. Interestingly, while MOPL30 supplementation led to increased EPA and DHA-rich
PC in plasma and RBC, the total level of PC decreased, with a concomitant increase in plasma
free choline. This may indicate a higher level of PC degradation as a result of increased dietary
intake, ensuring maintenance of the strictly regulated choline balance in the human body [41].
We were unable to measure acetylcholine in plasma due to its short half-life, however, both the
one-carbon cycle/remethylation process and the production of acetylcholine may potentially have
been stimulated by increased choline levels. DHA has been demonstrated to increase synaptic
transmission in mammalian brain cells, and this effect was potentiated by
phosphatidylcholine [42]. Thus, MOPL30 may have beneficial effects on cognitive function. It
would be valuable to measure plasma choline acetyltransferase activity in future clinical trials to
verify if acetylcholine production is stimulated by MOPL30 in humans.
As high plasma levels of long- and medium-chain acylcarnitines are linked to increased heart
failure in CVD patients, they have been put forward as potential biomarkers of cardiovascular
risk[13]. Incomplete -oxidation, impaired substrate switching, and dysregulation of
mitochondria during insulin resistance can cause elevated levels of intermediate oxidation
products, and this can be reflected in plasma acylcarnitine levels [43,44]. Thus, it is of interest to
establish whether dietary intervention with n-3 PUFAs affect these plasma parameters in healthy
adults, as EPA and DHA are known to stimulate mitochondrial -oxidation. We observed a
reduction in all acylcarnitines after a two-week intervention with MOPL30, including the riskassociated palmitoylcarnitine.
In further studies it will be interesting to determine if the supplement can benefit patients with
insulin resistance, both with regard to plasma TAG levels, mitochondrial function, and glucose
tolerance. It will be of particular interest to compare the bioactivity of MOPL30 and TAG EPA
and DHA supplements at lower doses of EPA and DHA. In a follow-up study, a double blind
comparison to fish oil will be performed to identify possible PL-specific effects of MOPL30.
Also, a rodent study is planned to examine bioaccretion of EPA and DHA into brain and other
tissues.
Conclusions
EPA and DHA-rich PC from herring roe was taken up by RBC during the two week intervention.
Several parameters in blood were affected, including a reduction in TAG and NEFA, and an
increase in HDL cholesterol, choline, and betaine. Further, there was improved glucose tolerance
among the participants after two weeks. Based on the findings from this short-term pilot study
with 2.4 g EPA + DHA per day, MOPL30 may provide significant effects on lipid status and
glucose tolerance.
Methods
Study subjects
This intervention study was performed at the University of Bergen according to Good Clinical
Practice Guidelines and the World Medical Association Declaration of Helsinki. The Regional
Ethics Committee, REK vest, approved the protocol (REK vest, approval no. 2013/112), and
informed consent was obtained from all the subjects.

Healthy young adults (21 women and one man) aged 21 to 26 years were recruited on a
voluntary basis. Of the 27 adults asked, six were not included in the study due to unwillingness
to take capsules and/or blood samples. Exclusion criteria were conditions requiring medication,
pregnancy and diabetes type I or II. The participants were instructed not to make any major
changes to their diet three weeks before, during, or four weeks after intervention, with the
exception of avoiding using fish egg products like caviar. Participants were instructed to perform
a three-day weighed food record within the two weeks before, as well as during the intervention.
Results were analyzed by the program Mat p Data
(http://www.matportalen.no/verktoy/mat_pa_data/ webcite), and mean dietary intake of nutrients
of interest were calculated as percentage of total energy intake (energy %).
Supplement and study design
MOPL30 is a capsulated herring roe PL supplement, where each capsule contains 511 mg total
lipid, of which 30% are PL, with 56 mg EPA, 158 mg DHA, and 12 mg n-3 DPA. The
participants received 11 capsules per day for 14 days, corresponding to a daily dose of 1738 mg
DHA and 616 mg EPA. Four capsules were taken at breakfast and lunch and three capsules were
taken at dinnertime. The last day of capsule intake (end), blood samples were drawn the next
morning between 8 and 11 am after an overnight fast, an oral glucose tolerance test was
performed (see description below), and capsules were divided between the remaining meals of
the day. Blood samples were drawn the next morning after taking the last capsule between 8 and
11 am after an overnight fast, followed by an oral glucose tolerance test. Of the 21 participants,
14 were recruited for additional fasting blood samples one week (WO week one) and four weeks
(WO week four) after the final day of supplement. One participant was excluded due to lack of
fasting at WO week four. All blood samples were centrifuged and EDTA-plasma and serum was
separated after a minimum of 15 minutes and maximum of 30 minutes at room temperature.
Blood samples for isolation of RBC were drawn in EDTA tubes, centrifuged at 3000 rpm for
10 minutes, and plasma and interface removed. RBC were subsequently washed three times in
PBS, with centrifugation and removal of buffy coat between each wash. All samples were
aliquoted and stored at 80C for further analysis.
Oral glucose tolerance test
After an overnight fast, blood was drawn for measurement of fasting glucose and insulin as
described above. In addition, a rapid analysis of blood glucose was performed using a FreeStyle
Lite (Abbott Diabetes Care, Inc., Alameda, CA, USA). Glucose dissolved in water was ingested
in no more than 5 minutes (300 ml 0.25 g/ml glucose with 5% lemon juice), and blood glucose
was measured by FreeStyle Lite at 10, 30, 60 and 120 minutes after glucose ingestion. The area
under the curve was calculated by Prism Graph-Pad Software (San Diego, CA, USA).
Enzymatic analysis of blood parameters
Lipids were measured enzymatically in EDTA plasma on a Hitachi 917 system (Roche
Diagnostics GmbH, Mannheim, Germany) using the triacylglycerol (GPO-PAP), cholesterol
(CHOD-PAP), HDL-cholesterol plus and LDL-cholesterol plus kit from Roche Diagnostics, and
the non-esterified fatty acid (NEFA FS) kit and the Phospholipids FS kit from DiaSys Diagnostic
Systems GmbH (Holzheim, Germany). Glucose was measured in EDTA-plasma using the
Gluco-quant Glucose/HK (GLU) kit from Roche Diagnostics. Insulin was measured using
routine methods at the central laboratory at Haukeland University Hospital.

Analysis of plasma total fatty acid composition, and plasma and RBC PC fatty acid
composition
The total fatty acid composition in EDTA-plasma was analyzed as previously described [45]. For
analysis of the plasma PC fatty acids, plasma total lipids were extracted based on Folch, then PC
was separated from other lipids by HPLC using YMC diol-120-NP column, 250 mm4.6 mm
ID, using hexane/acetone/methanol/chloroform (1/1/6/4) as the eluting solvent system. The
column effluent was spilt to an evaporative light scattering detector for quantitation and to
fraction collector for recovery. Fatty acids in the PC fraction were converted to their respective
methyl esters then separated and quantified by capillary column GLC [46].
Analysis of plasma and RBC lipid composition by 1H or 31P NMR
Lipids were extracted from 0.5 ml serum by Folch Solvent (1 ml each of CDCl3 MeOD and
CsEDTA (0.2 M), pH 8). After centrifugation, the lower layer was analyzed at 600 MHz cQNP
using a NMR spectrometer Avance III 600 (Bruker, Karlsruhe, D), magnetic flux density 14.1
Tesla, a QNP cryo probe, and automated sample changer Bruker B-ACS 120. Computer Intel
Core2 Duo 2.4 GHz under MS Windows XP and Bruker TopSpin 2.1 was used for acquisition,
while Bruker TopSpin 2.1 was used for processing [47-50].
Plasma choline, betaine, carnitine and acylcarnitines
Plasma choline, betaine, free carnitine and its precursors: trimethyllysine and -butyrobetaine, as
well as short-, medium-, and long-chain acylcarnitines, were analysed in plasma using
LC/MS/MS as described previously [10]. Stable isotope dilution LC/MS/MS was used for
quantification of choline and betaine. Choline and betaine were monitored in positive MRM MS
mode using characteristic precursor-product ion transitions: m/z 7658, m/z 10460
and m/z 11858, respectively. The internal standards, choline-trimethyl-d9 (d9-choline) and
d11-betaine, were added to plasma samples before protein precipitation, and were similarly
monitored in MRM mode at m/z 8566, m/z 11369 and m/z 12966, respectively.
Various concentrations of choline and betaine standards and a fixed amount of internal standards
were spiked into 4% albumin (BSA) to prepare the calibration curves for quantification of
plasma analytes.
Statistical analysis
Data was analyzed using Prism Software (Graph-Pad Software). The results are shown as means
with standard deviation (SD). DAgostino & Pearson omnibus normality test was used to
determine normal distribution. Paired t-test or Wilcoxon matched-pairs signed ranked test, for
parametric data and non-parametric data, respectively, were performed to evaluate statistical
differences between start and end samples, between end and WO week one, and between end and
WO week four samples. P-values<0.05 were considered significant.
Competing interests
This work was partly supported by Arctic Nutrition AS, and at the time of the study, AB was an
employee of Arctic Nutrition.
Authors contributions
BB, ES, JG, AB, and RKB planned and designed the study. BB, ES and JG performed the study.
PB performed the total plasma fatty acid composition assay. AS, BWKD, and SMI were
responsible for the acylcarnitine analysis, NMR plasma lipid composition analysis, and the

phosphatidyl choline fatty acid composition analysis, respectively. BB and AB performed

statistical analysis, analysed the data, and BB wrote the manuscript. All authors critically revised
the manuscript, and read and approved the final manuscript.
Acknowledgements
We would like to thank Kari Williams, Liv Kristine ysd, Randi Sandvik, and Roger A. Dyer
for excellent technical assistance. The University of Bergen through the Clinical Nutrition
Program, and the company Arctic Nutrition AS supported this work.
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